Fanconi anemia (FA) is a rare genetic disease characterized by physical birth defects, bone marrow failure, and cancer susceptibility. FA patient cells are hypersensitive to DNA crosslinking agents, e.g. mitomycin C (MMC). To date, thirteen FA genes have been identified, and the protein products of these genes function cooperatively in a pathway, the FA-BRCA pathway, to repair damaged DNA. Several FA patients exist, however, for whom the underlying genetic defect is unknown. Therefore, it can be concluded that new FA genes remain to be discovered. A critical step in the activation of the FA-BRCA pathway is the mono- ubiquitination of the Fanconi anemia D2 (FANCD2) protein. Mono-ubiquitination of the FANCD2 protein signals its translocation to the nucleus where it functions in DNA repair. The mono-ubiquitination of the FANCD2 protein is facilitated by the concerted activity of at least eight `upstream'FA core complex proteins. We have recently identified a Chinese Hamster Ovary mutant CHO UV-1, which has cellular characteristics indistinguishable from FA patient cells defective in any of the eight FA core complex components. For example, the CHO UV-1 cells are hypersensitive to MMC and are defective in the mono-ubiquitination of the FANCD2 protein. Furthermore, we have determined that the cellular defects of the CHO UV-1 cells can be rescued by the introduction of a fragment of human chromosome 11. These findings strongly suggest that a new FA-BRCA pathway gene resides in this chromosomal region. The goals of this proposal are to identify this gene. Two aims are proposed: First, using a bioinformatics approach we will analyze all the known genes in this region and compile a list of candidate FA-BRCA genes. Using RNA interference we will deplete these genes from normal cells and determine the effects on cellular sensitivity to MMC and on the mono- ubiquitination of FANCD2. Second, we will attempt to rescue the FA-like phenotypes of the CHO UV-1 cells by infection with a retroviral whole genome cDNA library. The infected CHO UV-1 cells will be grown in the presence of a pre-determined lethal concentration of MMC, and MMC-resistant clones will be isolated. The correcting cDNA will subsequently be recovered and identified. Our results could lead to the identification of a new FA-BRCA pathway gene on chromosome 11p. A greater understanding of the regulation of the FA-BRCA pathway will lead to improved diagnostic and therapeutics to FA, and improve our understanding of leukemia susceptibility in the FA and general (non-FA) populations. PUBLIC HEALTH RELEVANCE: A greater understanding of the regulation of the Fanconi anemia (FA)-BRCA pathway may lead to improved diagnostic and therapeutic approaches to FA, as well as improve our understanding of leukemia susceptibility in the FA and general (non-FA) populations.